Nosema Disease: Lack of Knowledge and Work ... - COLOSS

NOSEMA DISEASE: LACK OF KNOWLEDGE AND 

WORK STANDARDIZATION 

Scope of Workshop: 

Up to the moment, two different microsporidia species have been shown to infect Apis 

mellifera: Nosema apis and Nosema ceranae. While the disease due to N. apis has been widely 

studied, those due to N. ceranae only has been studied in the last four years. Due to the recent 

description of this last agent, there are several question about the epidemiology, pathology, 

diagnostic and control that have to bee answered. The workshop “Nosema disease: lack of 

knowledge and work standardization” has been designed to put all the knowledge together 

and to standardize some specific aspects for researching. The workshop goal will be to answer 

the most of questions that the researchers have nowadays or to determine how that questions 

could be studied to get answered. With this objective, all the COLOSS member will be asked 

for questions that feel have to be answered in an open call (even in the case they are not 

interested to join to the workshop). Questions will be grouped by topic and speakers will 

describe what it is know and unknown in every topic and a discussion will be made afterwards. 

Conclusions of Workshop: 

1. The disease caused by Nosema ceranae is not similar to nosemosis by Nosema apis. 

2. A proposal to differentiate nosemosis by Nosema apis as “nosemosis type A” and 

nosemosis by Nosema ceranae as “nosemosis type C”. 

3. Koch´s postulates have been confirmed in nosemosis by Nosema ceranae. 

4. There is a relationship between Nosema ceranae and bee losses in some cases. 

5. Standard protocol for experimental infection will be discussed by a specialist panel that 

has been formed during the Workshop: Marie Pierre Chauzat (Head), Violeta Santrac, 

Zachary Huang, Asli Özkirim, Raquel Martín-Hernández, Ulrike Hartmann, Claudia 

Dussabaut, Frederic Delbac, Antonio Nanetti. 

6. There is a need of Nosema apis genome to compare both (N. apis and N. ceranae) to 

find differences that can explain observations 

7. New genetic markers must be developed for monitoring the disease

8. New polymorphic markers are needed to define an isolate (to differentiate genotypes or 

strains). 

9. Nosema ceranae shows different epidemiological patterns in Europe. 

10. Management practices and beekeeping material have an important role in transmission. 

11. Nosema ceranae is considered as an emerging pathogen in honeybee in the 21st century 

and can be found in other non-apis insects. 

12. For Nosema apis, spore counts and clinical signs are considered as the best available 

diagnosis. 

13. The development of standard protocol for sampling and diagnosis of nosemosis by 

Nosema ceranae will be discussed by a specialist panel that has been formed during the 

Workshop: Antonio Nanetti, Zachary Huang, Giovanni Formato, Mariano Higes, Anna 

Gajda, Marie-Pierre Chauzat, Asli Özkirim and Martin Kamler. 

14. Nosemosis by Nosema ceranae can be controlled by fumagillin, but EU regulatory 

restraints are present. Furthermore, differences in fumagillin stability according to 

preparation, administration, conservation conditions may affect residues and efficacy. 

15. There are some “alternative” possibilities to limit the development of Nosema ceranae 

with “natural” products (meeting requirements of organic beekeeping). Residue studies 

are required for consumer safety. 

16. Beekeeping management such as renewal of combs or queens may play an important 

role in controlling nosemosis.

Time 

Workshop Program 

19.10.2009 (Monday) 

Arrival and Hotel accommodation in Guadalajara city. 

20:00 Social event 

20.10.2009 (Tuesday) 

9:00-9:30 Registration 

9:30-10:00 Official Welcome and presentations. 

10:00-10:30 Plenary talks: Honeybee nosemosis. Disease or syndrome 

Dr. Mariano Higes, Raquel Martín, Researcher from the Apicultural Center (CAR), JCCM, Spain 

10:30-11:00 Coffee break 

11:00-12:00 Plenary talks about aetiology and pathology: 

1. How does Nosema apis make honey bees forage earlier Dr. Zachary Huang. Associate Professor, Michigan State 

University, USA. 

2. Physiological and behavioural changes in Nosema infected bees: A model to understand colony collapse. Dr. Dhruba 

Naug. Assistant professor Colorado State University, USA. 

3. Histopathology of Nosema infected bees. Aránzazu Meana. Associate professor of Pathology, Veterinary Faculty, 

Complutense University of Madrid. Spain. 

12:00-13:30 Presentations and Q&A about aetiology and pathology.. 

- Preliminary results on cage experimentations of adult honey bees fed with spores of N. ceranae. Chauzat MP. 

- Nosema spp. infection in Spain: Consequences in colony productivity and vitality. Cristina Botías. 

- Interactive effects between Nosema microspores and a neonicotinoid in honeybees. Dussaubat, C. 

- To bee or not to bee: differential mortality induced by Nosema ceranae Ulrike Hartmann. 

Open discussion 

14:00-15:30 Lunch 

15:30-16:30 Plenary talks about Nosema genome and molecular detection. 

1. A comparative genomic approach to the study of microsporidian parasite, Nosema. Judy Chen, Research Entomologist. 

Agricultural Research Services, United States Department of Agriculture, USA. 

2. Diversity and recombination of rDNA in the microsporidian Nosema ceranae: how reliable is the genotyping Nuno 

Henriques-Gil, Genetics Laboratory, San Pablo-CEU University, Madrid. Spain. 


17:00-18:00 Presentations and Q&A about Nosema genome and molecular detection. 

- Molecular basis of genome interaction of the honeybee Apis mellifera with an evolutionary old and novel 

introduced Nosema species. Matthias Müller. 

- Genetic variation in resistance to Nosema infection within honeybee colonies. Katherine Roberts. 

- New tools for epidemiological and pathogenicity studies of Nosema ceranae. Frédéric Delbac. 

- Molecular diagnosis of Nosema – what’s the limit of detection S. Erler. 

Open discussion. 

20:30-Open Social Dinner 

21.10.2009 (Wednesday) 

9:00-10:00 Plenary talks about control (epidemiology and diagnostic) 

1. Epidemiology of Nosema ceranae. Dr. Aránzazu Meana, Associate professor of Animal Health, Veterinary Faculty, 

Complutense University of Madrid. Spain. 

2. Epidemiology of Nosema ceranae in the Netherlands. Methodology and Complexities. Romee Van der Zee. Head of 

Netherlands Centre for Bee Research, NCB. Netherland. 

3. Nosema Diagnostic. Dr. Raquel Martín, Researcher from the Apicultural Center (CAR), JCCM, Spain 

10:00-11:00 Pesentations and Q&A about epidemiology and diagnostic. 

- Nosema situation in the Czech Republic. Martin Kamler. 

- Incidence of Nosema spp. and colony performance in Austria 2006–2008. Derakhshifar, I.. 

- Presence of Nosema apis and Nosema ceranae in Italian apiaries. Franco Mutinelli. 

- First detection of Nosema ceranae in Apis mellifera from Bosnia and Herzegovina. Violeta Santrac 

- Two Diagnostic Methods of Nosemosis in Turkey. Aygun Yalçınkaya. 

- The size of bee sample for investigation of Nosema sp. infection level in honey bee colony. Anna Gajda. 

- Parasite infections of pollinator communities. Sophie Evison. 



11:30-12:30 Plenary talks about control (treatment and profilaxis) 

1. ApiHerb as an alternative product to treat Nosema infection. Dr. Antonio Nanetti. Researcher, CRA- Unità di Ricerca di 

Apicoltura e Bachicoltura. Bologna. Italy 

2. Fumagillin stability. Dr. Jose Luis Bernal, Professor of Analytical Chemistry. Faculty of Chemistry, Valladolid 

University. Spain 

3. Thymol: an alternative treatment for control of Nosema ceranae Dr. Cecilia Costa. Researcher, CRA- Unità di Ricerca 

di Apicoltura e Bachicoltura. Bologna. Italy 

12:30-13:30 Presentations and Q&A about treatment and profilaxis. 

- Medication possibilities against new age-nosemosis. Békési László, Szalai Enik. 

- 

Potential of microalgae and plant extracts for the control of nosemosis in honeybees. Frédéric Delbac. 

- Effectiveness in reducing the number of Nosema spores of Api Herb and Vita Feed Gold. Giovanni Formato. 

- Epidemyology and Treatment of Nosemosis in Turkey. Aslı Özkırım. 

- Rare Nosema infections in Denmark. Per Kryger 


13:30-14:00 Elaboration of conclusions 

14:00-15:30 Lunch and end of workshop 

20:30-Open Social Dinner - Optional only for those interested to stay one day more for practical work in Centro Apícola Regional (CAR) 

22.10.2009 (Thursday) Optional 

9:00-9:30 Transport Hotel to Centro Apícola Regional (CAR) 

9:30-14:00 Practical work in field and laboratory 

13:00-14:00 Lunch and end of workshop

List of participants 

1. Martin Kamler, MVD 

Department of Parasitology, University of Veterinary and Pharmaceutical Sciences. 

Palackeho 1/3, Brno 612 42, Czech Republic 

+420 541 562 270, +420 605 941 987 

martan79@yahoo.com 

2. Anna Gajda, DVM 

Warsaw University of Life Sciences, Ciszewskiego 8 Street 

Warsaw 02-786, Poland 

+48(022)5936140, +48518111938 

anna_gajda@sggw.pl 

3. Marie-Pierre Chauzat, Dr 

French Food Safety Agency (AFSSA) 

105, route des Chappes. BP 111. Sophia Antipolis cedex 

06 902, Frannce 

00 33 4 92 94 37 21, 00 33 6 85 90 78 42 

mp.chauzat@afssa.fr 

4. Asli Ozkirim, Dr. 

Hacettepe University, Hacettepe Univ. Department Of Biology Bee Health Lab. 06800 Ankara, Turkey 

0090 312 297 80 43, 0090 533 326 97 13 

ozkirim@hacettepe.edu.tr 

5. Giovanni Formato, DVM 

Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova, 1411 – 00178 Roma, Italy 

+39.06.790991 (office), +39.349.5330816 

gioformato@yahoo.es 

6. Ulrike Hartmann 

Swiss Bee Research Centre, Schwarzenburgstrasse 161 

Bern 3003, Switzerland 

+41 31 324 74 24 

hartmann.ulrike@alp.admin.ch 

7. Franco Mutinelli, Dvm, 

Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università, 10, Legnaro (Padova) 35020, Italy 

0039 049 8084287, 0039 348 4405586 

fmutinelli@izsvenezie.it 

8. Aygun Yalcinkaya, 

Research Assistant, Hacettepe University, Dept. Of Biology, Bee Health Lab. Beytepe Campus, Ankara 06800, Turkey 

+903122978011 +905367869321 

aygun@hacettepe.edu.tr 

9. Sophie Evison, Dr 

University of Leeds, Institute of Integrative and Comparative Biology, Miall Building, University of Leeds, Leeds LS2 9JT, UK 

+44 (0)113 3437214 

+44 (0)7740 865295

w.o.h.hughes@leeds.ac.uk 

10. Irmgard Derakhshifar, Dr. 

Austrian Agency for Health and Food Safety, Institute for Apiculture, Spargelfeldstraße 191, Vienna 1220, Austria 

+43 (0) 50 555-33122 

irmgard.derakhshifar@ages.at 

11. Laszlo Bekesi, Dr. 

Res. Inst. For Animal Breeding and Nutrition, Gesztenyés u. 1., Herceghalom 2053, Hungary 

36-23-319-133 36-20-411-0123 

bekesi@katki.hu 

12. Katherine Roberts, Miss 

University of Leeds, Institute of Integrative and Comparative Biology, Miall Building, University of Leeds, Leeds LS2 9JT, UK 

+44 (0)113 3437214, +44 (0)7793 963002 

bskr@leeds.ac.uk 

13. Claudia Dussaubat Arriagada, PhD student 

INRA Avignon, Biology and Honey Bee Protection Laboratory, UMR 406, Abeilles et Environnement, Site Agroparc - Domaine Saint Paul, 

84914 Avignon Cedex 9, Avignon 84914, France 

(33) (0)4 32 72 26 01, 06 70 31 82 25 

cdussaubat@avignon.inra.fr 

14. Judy (Yanping) Chen, Dr. INVITED EXPERT 

Research Entomologist. Agricultural Research Services, United States Department of Agriculture, 10300 Baltimore Avenue, Bldg 476 Barc- 

East, Beltsville, Md, 20705 

(301) 504-8749 (301) 504-8736 

Judy.Chen@ars.usda.gov 

15. Zachary Huang. Dr. INVITED EXPERT 

Associate Professor, Michigan State University. 243 Natural Science, Dept of Entomology, East Lansing, Michigan 48824, USA 

517-353-8136, 517-980-1200 

bees@msu.edu 

16. Dhruba Naug, Dr. INVITED EXPERT 

Assistant Professor, Colorado State University, 1878 Campus Delivery, Fort Collins, CO 80523, U.S.A. 

970 491 2651 

dhruba.naug@colostate.edu 

17. Romee Van der Zee INVITED SPEAKER 

Head of Netherlands Centre for Bee Research, NCB. Durk Dijkstrastr. 10 9014 cc Tersoal, Netherland. 

romee@van.der.zee@beemonitoring.org 

18. Cecilia Costa, Dr. INVITED SPEAKER 

CRA – Research Unit of Apiculture and Sericulture. Via Fratelli Rosselli 80, Reggio Emilia, I – 42100, Italy 

+39 0522 285532 

cecilia.costa@entecra.it 

19. Antonio Nanetti, Dr INVITED SPEAKER 

CRA – Research Unit of Apiculture and Sericulture. Via di Saliceto 80, Bologna 

I – 40128, Italy 

+39 051 353103 +39 329 1857975

antonio.nanetti@entecra.it 

20. Violeta Santrac 

Veterinary Institute RS Branka Radicevica 18 78000 Banja Luka Bosnia and Herzegovinia, 

vsantrac@yahoo.com 

21. Silvio Erler, Dipl.Bio. 

Martin-Luther-Universität Halle-Wittenberg, Institut für Biologie-Zoologie, Molekulare Ökologie, Hoher Weg 4, Halle 06099, Germany 

+49-3455526235 

silvio.erler@zoologie.uni-halle.de 

22. Mattias Müller, Diplom Biologe 

Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 4, Halle 06120, Germany 

0049 345 55 26 398 0049 176 2980 3946 

matthias.mueller@sfi.uni-halle.de 

23. Eniko Szalainé Mátray, Dr. 

Institute for Animal Research, Csanak u. 11, Gödöllö, H-2100, Hungary 

+36 28 511 344 +36 30 66 44 458 

matray@katki.hu 

24. Frédéric Delbac, Dr 

Professor in Microbiology - Université Blaise Pascal, Laboratoire Microorganismes : Génome et Environnement – UMR CNRS 6023 –Equipe 

Interactions Hôtes-Parasites. 

24 Avenue des Landais, Aubiere Cedex 63177, France 

(33)(0)4.73.40.78.68 (33)(0)6.65.72.38.04 

frederic.delbac@univ-bpclermont.fr 

25. Per Kryger, Dr 

University of Aarhus, Research Centre Flakkebjerg, Slagelse 4200, Denmark 

+4589993629 +4522283329 

Per.kryger@agrsci.dk 

ORGANIZERS AND SPANISH COLLABORATORS 

26. Mariano Higes 

27. Raquel Martín 

Researchers from the Apicultural Center (CAR), JCCM, Spain 

+34.949.25.00.26 +34.949.25.01.76 

rmhernandez@jccm.es 

mhiges@jccm.es 

28. Aránzazu Meana 

Dr., Associate professor of Animal Health, Veterinary Faculty, Complutense University of Madrid. Avda. Puerta de Hierro s/n, 28040 Madrid, 

Spain. 

+34.91.394.39.03 

ameana@vet.ucm.es 

29. Nuno Henriques-Gil, Dr. 

Genetics Laboratory, San Pablo-CEU University, Urbanización Montepríncipe, 

28668 Boadilla del Monte, Madrid, Spain. 

nhengil@ceu.es

30. Jose Luís Bernal, Dr 

Professor of Analytical Chemistry. Faculty of Chemistry, Valladolid University. Spain 

jlbernal@qa.uva.es 

31. Amparo Martínez; Dr 

Epidemiologist. TRAGSEGA, Julián Camarillo 6-A, 4 planta sector D, 28037 Madrid. 

amarti13@tragsa.es

COST Action FA0803 - Prevention of honeybee COlony LOSSes 

Nosema disease: lack of knowledge and work standardization 

Abstract Submittal Form 

Title: Please limit your title to 150 characters. 

Two Diagnostic Methods of Nosemosis in Turkey 

Authors and Affiliations: Please list all authors and their affiliations. For the contact 

author only (typically the senior author), include email address, mailing address, 

and phone number. Denote contact author with an asterisk = *. 

Aygun Yalçınkaya, Nevin Keskin, Aslı Özkırım* 

Hacettepe University Department of Biology Bee Health Laboratory 06800 Beytepe- 

Ankara/TURKEY 

Author of correspondance: ozkirim@hacettepe.edu.tr 

Text of Abstract: Please limit abstract text to 250 words. 

Nosemosis is a common honeybee disease in Turkey. Generally, honeybee gut is used for 

diagnosis of Nosemosis in honeybees. In some laboratories, honeybee gut is taken out 

and put on slides. After that it is diluted by saline solution and investigated for occurrence 

of Nosema spp. spores under the light microscope. By this method, it is not considered 

the infection level. So, It couldn’t be possible to apply proflactic treatment of colonies. 

Other laboratories not only detect Nosema spp. Spores, but also infection level in Turkey. 

In our laboratory, all samples from different regions of Turkey are collected in spring and 

autumun. They are registered and classified according to the local region. At least 30 

honeybee samples from each colonies are collected. Honeybees’ gut are taken out via 

forceps and collected in a dish then diluted 1 ml of saline solution per bee. 0.1 ml of the 

solution are put on heamocytometer and counted Nosema spp. spores under the light 

microscope. It is calculated the number of Nosema spores per bee. By this method, it is 

possible to determine infection level of Nosemosis. Because it is also known that 

infection level is the most important thing for evaluating the distribution of Nosema 

disease and the kind of treatment of Nosemosis. 

In order to distinguish the species of Nosema (apis/cerena), molecular methods have 

been just applied in one laboratory in Turkey, but there isn’t any publication about this 

research yet.





Medication possibilities against new age-nosemosis 




1 László, Békési; 1 Enik, Szalai Mátray; 1 Lívia, Harka; 2 Dénes, Hegeds; 3 Attila, Albert 

1 Research Institute for Animal Breeding and Nutrition, H-2101 Gödöll 

2 

MgSzH Veterinary Directorate of Bács-Kiskun County, Kecskemét 

3 ANIVET Kft., Budapest 

Corresponding authors address: bekesi@katki.hu 


Though nosemosis caused by Nosema apis has been a well known bee disease for a long 

time, questions of the prevention and control are intensively discussed by beekeepers all 

over the world again. The uncertainty of its epidemiology increases the anxiety induced 

both by colony collapse disorder in many places, and by the presence of the newly 

introduced species N. ceranae. In our investigations the efficacy of the well known 

fumagillin antibiotics has been compared with another preparation of different structure 

(Nonosz®.) 

Although the registration of fumagillin has been withdrawn in EU countries a great many 

beekeepers are using it all over the world. 

Since the utilization of antibiotics has more and more limitation in food stuff producing 

animals, finding an appropriate (non-antibiotic) replacement is a recurring problem. 

Nonosz® is a Hungarian made preparation of curative effect (sodium ortho-hydroxycarbonic 

acid and Beta vulgaris cv.- Chemor Kft) that entered into the market on the basis 

of preliminary positive results against nosemosis. 

Both in laboratory induced infections and in experiments on infected honeybee colonies 

the efficacy of the two products gave similar results in decreasing the spore production. 

According to the results of the laboratory tests with N. ceranae delayed effect of the 

Nonosz was detected. Some loss occurred in the first days, but the spore production 

remained much below the control. 

Our experiments seem to be evidence about the efficacy of fumagillin against N. ceranae 

and also demonstrating that alternatives may exist for the medication of new-age 

nosemosis.





Fumagillin stability in different media 




J. L.Bernal*, M. J.Nozal, L.Toribio, M. T. Martín. José Bernal. 

Analytical Chemistry. Faculty of Sciences. University of Valladolid. 47011 Valladolid, 

Spain. 

*Professor of Analytical Chemistry, jlbernal@qa.uva.es, Faculty of Sciences.University 

of Valladolid. 47011.Valladolid.Spain, phone:34 983 423280 


The stability while using a commercial formulation, Fumidil B, which contains 

Fumagillin dicyclohexilamine, is affected by several factors. The water to dissolve the 

product has some influence so it must be neutral and of mild hardness. Fresh solutions 

stored in amber containers and kept in a fridge can hold out for a month. The use of syrup 

or honey-sugar patty, as a medium to apply the product, favours the stability of the 

compound. Temperature has a slow effect on the degradation, whereas sunlight or UV 

exposure reduces drastically, in a few hours, the initial concentration of fumagillin. 

Laboratory tests suggest that the mixture of honey-powdered sugar is the best option to 

apply the formulation.





NOSEMA SPP. INFECTION IN SPAIN: CONSEQUENCES IN COLONY 

PRODUCTIVITY AND VITALITY 




C. Botías*, R. Martín-Hernández*, A. Meana**, M. Higes* 

*Centro Apícola Regional. Camino de San Martín s/n, 19180 Marchamalo, Spain. 

** Veterinary Faculty, Complutense Unversity of Madrid, Avda. Puerta de Hierro s/n, 

28040 Madrid, Spain. 

Corresponding author: M. Higes, mhiges@jccm.es 


During the last years, Nosemosis has shown to be an increasing beekeeping problem 

worldwide. Nowadays, two different Nosema species have been identified in Apis 

mellifera: Nosema apis and Nosema ceranae. 

A long asymptomatic incubation period in the N. ceranae disease has been described 

(Higes et al., 2008), involving continuous death of adult bees, non-stop brood rearing by 

bees and colony loss in winter or early spring despite the presence of sufficient remaining 

pollen and honey. 

Evolution of 50 naturally infected colonies of Apis mellifera iberiensis during one year is 

reported. In September 2007 the 50 colonies were infected by N. ceranae, and 26 of them 

were infected by N. apis too (PCR tested). The colonies were divided into 5 groups of 10 

colonies each: (C) Control (5 unmanaged colonies); (CS) Control with Syrup (5 colonies 

supplied with syrup every season); (1T) One treatment in a year (5 colonies provided 

with Fumagillin in Autumn); (2T) Two treatments in a year (5 colonies treated with 

Fumagillin in Autumn and Spring); (4T) Four treatments in a year (5 colonies provided 

with Fumagillin every season). 

The group of colonies with 2 treatments in a year was the most productive (5 times more 

honey than the control group unmanaged) and showed a high level of vitality, considering 

the amount of brood cells and the population of adult bees as a sign of it. The group with 

4 treatments showed similar results. 

The control groups, in spite of being asymptomatic, were less productive and less 

populated. The unmanaged control colonies registered the highest number of dead 

colonies.





Preliminary results on cage experimentations of adult honey bees fed with spores of 

N. ceranae 




Chauzat MP, Ribière-Chabert M, Villier A, Schurr F, Blanchard P, Bouveret C, 

Drajnudel P and Faucon JP 

French Food Safety Agency (AFSSA), 105 route des Chappes, BP 111, 06 902 Sophia 

Antipolis France. 


Infections with Nosema apis spores have been largely reported in the literature. A new 

species of Nosema has been recently identified on the honey bees. Caged experiments 

have shown high and rapid mortality of adult bees when they were fed with spores of 

Nosema ceranae. 

Adult honey bees have been caged in order to study the mortality induced by the feeding 

of syrup supplemented with Nosema ceranae spores collected in France. 

Various factors have been studied in order to better define the optimum experimental 

conditions : spores conservation, number of spores in the inoculums, age of honey bees, 

quality of food given to caged insects, temperature. 

Results will be given en multiplication on spores and mortality of honey bees. 

Experimental conditions will be discussed.





A comparative genomic approach to the study of microsporidian parasite, Nosema 




Yanping (Judy) Chen 1 , R. Scott Cornman 1 , Yan Zhao 2 , Jeffery S. Pettis 1 and Jay D. 

Evans 1 , 

1 USDA-ARS Bee Research Laboratory, Beltsville, MD 20705, 

2 USDA-ARS Molecular Plant Pathology Laboratory, Beltsville, MD, 20705. 


Nosema ceranae, originally considered a microsporidian parasite of Eastern 

honey bees, Apis cerana, is, along with the long known resident species, N. apis, a 

disease agent in European honey bees, A. mellifera. In order to gain more insight into the 

epidemiology, pathogenicity, and evolution of Nosema parasitism in bees, we have used 

a genomic approach to determine 1) the historical occurrence of two Nosema species in 

both honey bee hosts, 2) the tissue tropism, secondary structures of rRNA, and 

phylogenesis of two Nosema species, 3) the complete sequences of the N. ceranae 

genome and nearly completed sequences of N. apis genome. Our results showed that both 

Nosema species produced single and mixed infections in European and Asian honey bees 

and that N. ceranae is much more invasive in both host species than N. apis. While 

ultrastructural features showed that both species possess all of the characteristics of the 

genus Nosema, the tissue tropism was species specific which may lead to the difference 

of two Nosema species in host pathogenicity. The 454 pyrosequence of N. ceranae lead 

to a draft assembly (7.86 MB) and annotated genome, showing that the genome was 

highly AT-biased. Of 2,614 predicted protein-coding sequences, the genes conserved 

among microsporidia lack clear homology outside this group. Future comparisons of the 

genes conserved among microsporidia in two Nosema species will provide valuable 

insights for identifying virulence factors of parasites and in turn should translate into new 

strategies for diminishing the effects of parasites and improving honey bee health.





Interactive effects between Nosema microspores and a neonicotinoid in honeybees 




Alaux C., Dussaubat, C., Brunet J-L., Mondet F., Tchamitchan S., Cousin M., Brillard J., 

Baldy A., Belzunces L.P., Le Conte Y*. 

INRA, UMR 406 Abeilles et Environnement, Avignon 

Contact author : 

leconte@avignon.inra.fr 

INRA, UMR 406 Abeilles et Environnement 

Site Agroparc, 84914, Avignon, France 

Phone : + 33 04 32 72 26 27 


In the past years honeybee decline has been reported almost worldwide. Many single 

factors like parasites and pesticides have been identified as a potential cause; however, a 

combination of these agents is more likely to contribute to honeybee losses. 

Consequently, we tested the hypotheses describing honeybee losses as a multifactorial 

syndrome by investigating the interactive effects of Nosema microspores and an 

insecticide neonicotinoid on honeybee health in laboratory and field conditions. In order 

to measure mortality and energetic demands during the first 10 days of honeybee life, 

honeybees were artificially infected with 200,000 spores/bee of Nosema, and held in 

laboratory conditions. Then, they were exposed chronically to the neonicotinoid at three 

different concentrations encountered in nature. In parallel, immune parameters were 

studied in bees infected with the same Nosema dose in combination with one dose of the 

neonicotinoid. Furthermore, a similar experiment was conducted in the field over 30 

days. Mortality and foraging behavior were recorded. In addition, spore transfer between 

infected and non-infected honeybees within hive was studied. Concerning the laboratory 

experiment, the results of honeybee mortality and energetic demands will be shown.





Thymol: an alternative treatment for control of Nosema ceranae 




Cecilia Costa 1 *, Marco Lodesani 1 , Lara Maistrello 2 , Francesco Leonardi 2 , Franco 

Mutinelli 3 , Anna Granato 3 

1 Consiglio per la ricerca e la sperimentazione in apicoltura – Unità di ricerca di 

apicoltura e bachicoltura (CRA-API). 

2 Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio 

Emilia, via G. Amendola 2, Area San Lazzaro – Pad. Besta, 42100 Reggio Emilia, Italy 

3 Istituto Zooprofilattico Sperimentale delle Venezie, viale dell'Università, 10, 35020 

Legnaro (Padova) Italy 

* cecilia.costa@entecra.it 

Via di Saliceto, 80 – 42100 – Bologna (Italy) 

Tel. +39 051 353103 


In a first series of trials the natural compounds thymol, resveratrol, vetiver essential oil 

and lysozyme were assessed for potential use in control of Nosema ceranae infection of 

honey bees. None of the substances showed an increased bee mortality or decreased 

dietary preference. Adult worker bees from a nosema-free apiary were individually 

infected with 1 l of sucrose syrup containing 18000 N. ceranae spores, placed in cages 

and fed with candies cpntaining the screened substances. Infection levels were monitored 

over 25 days, by removal and dissection of 2 live bees per cage. On day 25 post-infection 

bees fed with candies containing thymol and resveratrol had significantly lower infection 

rates, and bees supplied with resveratrol candy also lived significantly longer. 

In a second set of trials the two most promising active ingredients, thymol (100 ppm) and 

resveratrol (10 ppm), were supplied to artificially infected bees in candy or in syrup, with 

the same procedure used in the first trials. On day 25 post-infection bees fed with thymol 

syrup had significantly lower levels of infection compared to control bees. Bees fed with 

thymol and resveratrol syrup lived significantly longer than bees fed with control syrup. 

Following the promising results, field tests were performed by feeding naturally nosema 

infected hives with syrup containing thymol during two consecutive springs. In the first 

year no significant differences in infection levels were observed, while in the second year 

the hives treated with thymol had a significantly greater decrease in infection compared 

to the control group.





Potential of microalgae and plant extracts for the control of nosemosis in honeybees 




DELBAC Frédéric*, POINCLOUX Delphine, SAUTEL François, DIOGON Marie, 

VIDAU Cyril, TEXIER Catherine, FONTBONNE Régis, VIVARES Christian, BLOT 

Nicolas, AUSSEIL Frédéric, EL ALAOUI Hicham 

Pr Frédéric DELBAC 

Laboratoire "Microorganismes : Génome et Environnement" 

UMR CNRS 6023 - Université Blaise Pascal 

24 Avenue des Landais, 63177 Aubière cedex 

FRANCE 

Tel: (33)(0)4.73.40.78.68 ; Fax: (33)(0)4.73.40.76.70 

e-mail: frederic.delbac@univ-bpclermont.fr 


Nosemosis is one of the most frequently observed parasitic pathologies affecting adult 

honeybees. This disease is due to Microsporidia, some obligate intracellular fungi-related 

parasites. Nosema apis was the historically described microsporidian species in Apis 

mellifera. This species can invade and proliferate in the ventriculus and midgut epithelial 

cells causing digestive disorders including diarrhea. More recently another species named 

Nosema ceranae has been also shown to colonize honeybees and seems to be now the 

predominant microsporidian infection in A. mellifera. The treatment of Nosema diseases 

was long-time done using Fumidil-B, a water soluble form of fumagillin. However, as 

this antibiotic is no longer licensed in the majority of EU member states, new molecules 

need to be identified. The aim of our study is to screen food additives like microalgae and 

plant extracts to control the development and spread of Nosema parasites and thus 

prevent this infectious disease. As no in vitro culture was available for both N. apis and 

N. ceranae, the efficacy of the natural extracts has been first investigated by ELISA using 

human cells infected by the species Encephalitozoon cuniculi as microsporidian model. 

Antiparasitic activity of more than 300 microalgae and 2200 plant extracts (collaboration 

with UMR CNRS-Pierre Fabre, Toulouse, France) has been evaluated. Currently, 20 

extracts showed a 80% inhibition of parasite growth using this in vitro drug screening 

assay. The next step will consist in the validation of these in vitro effective extracts in 

experimentally infected caged-bees.





New tools for epidemiological and pathogenicity studies of Nosema ceranae 




DELBAC Frédéric*, POINCLOUX Delphine, DIOGON Marie, VIDAU Cyril, TEXIER 

Catherine, FONTBONNE Régis, VIVARES Christian, BLOT Nicolas, EL ALAOUI 

Hicham 

Pr Frédéric DELBAC 

Laboratoire "Microorganismes : Génome et Environnement" 

UMR CNRS 6023 - Université Blaise Pascal 

24 Avenue des Landais, 63177 Aubière cedex 

FRANCE 

Tel: (33)(0)4.73.40.78.68 ; Fax: (33)(0)4.73.40.76.70 

e-mail: frederic.delbac@univ-bpclermont.fr 


Two microsporidian species were characterized in Apis mellifera: Nosema apis and the 

more recently described Nosema ceranae. Spores of both species can be obtained from 

naturally and/or experimentally infected bees. Our main objectives are to study the 

epidemiology of both species and to better understand the host-parasite interactions. 

Thus, we are developing some culture models for the in vitro proliferation of these 

microsporidian species. Preliminary results indicate that human fibroblasts could be 

useful for propagation of N. ceranae. 

Another project concerns the identification of new markers as tools for a more precise 

molecular epidemiology. We recently selected some genes coding for potential polar tube 

(PTPs) and spore wall (SWPs) proteins in the genome database of N. ceranae. Such 

components have been previously demonstrated to be interesting markers for mammal 

microsporidiosis diagnostic. Analysis of the genetic diversity of these markers in N. apis 

and in different N. apis and N. ceranae strains will be then undertaken. In order to raise 

specific antibodies we also recently produced some N. ceranae PTPs and SWPs in 

Escherichia coli and injected these recombinant proteins in mice. Some comparative 

genomic and proteomic approaches will help us to characterize microsporidia-specific 

protein coding genes that could be related to pathogenicity and to elucidate some 

metabolic pathways that could be potential targets for anti-Nosema drugs.





Incidence of Nosema spp. and colony performance in Austria 2006–2008 




Derakhshifar I.*, Köglberger, H., Oberlerchner, J., Moosbeckhofer R.* 

*AGES, Institute for Apiculture, Spargelfeldstraße 191, 1220 Vienna; 

irmgard.derakhshifar@ages.at; + 43 (0) 50555-33122 


During the years 2006 to 2008 465 dead bee samples from dead or weakened colonies or 

from colonies with noticeable bee mortality or colonies with suspected poisoning were 

investigated for Nosema infection. By light microscopy 85 samples (=18 %) were rated 

as Nosema spore positive in total. Bees from the group with suspected poisoning had the 

highest Nosema spp. incidence (37 %), followed by samples collected from spring to 

autumn (18 %) and during the winter period (16 %), respectively. 

Nosema incidence (%) in bee samples from (A) winter period and from (B) spring to 

autumn was considerably higher in dead than in living colonies (A: dead colonies: 21 %; 

living colonies: 13 %; B: dead colonies: 22 %; living colonies: 14 %, respectively). These 

results indicate that an infestation by Nosema spp. increases colony mortality during the 

winter period and the active season. 

The contrary was the case in bee samples from colonies with suspected poisoning (dead 

colonies: 29 %; living colonies: 38 %). This could mean that the higher losses of colonies 

were partly caused by other factors than Nosema. 

For species differentiation 126 samples from the total pool were analyzed subsequently 

by PCR. 67 of these samples (53 %) were tested negative for Nosema and 59 samples 

positive (47 %). N. ceranae was confirmed in 30 % of the positive samples and N. apis in 

10 %. 6 % of samples were infested by both Nosema species. 

Both Nosema-species were present in all federal provinces of Austria, except in 

Vorarlberg (adjacent to Switzerland), where only N. ceranae was detected.





Molecular diagnosis of Nosema – what’s the limit of detection 




S. Erler & H.M.G. Lattorff 

Dipl.-Bio. Silvio Erler * 

Martin-Luther-Universität Halle-Wittenberg, Naturwissenschaftliche Fakultät I – 

Biowissenschaften, Institut für Biologie, Institutsbereich: Zoologie 

AG: molekulare Ökologie, Hoher Weg 4, 06099 Halle (Saale), Germany 

Phone: ++49-345-5526235 

FAX: ++49-345-5527264 

Mail: silvio.erler@zoologie.uni-halle.de 

Dr. Michael Lattorff 

Martin-Luther-Universität Halle-Wittenberg, Naturwissenschaftliche Fakultät I – 

Biowissenschaften, Institut für Biologie, Institutsbereich: Zoologie 

AG: molekulare Ökologie, Hoher Weg 4, 06099 Halle (Saale), 

Germany 

phone. +49-345-5526389 

fax. +49-345-5527264 

email. lattorff@zoologie.uni-halle.de 


During last years different methods for the diagnosis of Nosema disease in bees have 

been developed. Molecular tools might have a strong advantage compared to microscopic 

methods – higher sensitivity. However, currently very little is known about the limit of 

detection of different molecular methods. The limits of these methods strongly depend on 

sample preparation and assay development. The evaluation of detection limits might 

influence the outcome of molecular diagnostics, especially as the rate of false negatives 

might be influenced. 

Here we present a comparative study on Nosema detection methods in different bees 

(honey bees and bumble bees) focussing on the detection limits. Different published and 

unpublished primers for PCR based screening methods were analysed for their sensitivity 

in molecular assays utilizing three different Nosema species (N. apis, N. ceranae and N. 

bombi). 

We found high differences in detection levels for the different Nosema diagnosis methods 

depending on the Nosema species and the primers based on the similar spore loads and 

dilution series.





Parasite infections of pollinator communities 




Sophie Evison 1 , Katherine Roberts 1 , Jacobus Biesmeijer 1 , Judith Smith 1 , Giles Budge 2 & 

William Hughes 1* 

1 

University of Leeds 

2 The Food and Environment Research Agency 

* Author for correspondence: 

Institute if Integrative and Comparative Biology 

Faculty of Biological Sciences 

Miall Building 


Leeds 

LS2 9JT 

UK 

Phone: +44 (0)113 3437214; Email: w.o.h.hughes@leeds.ac.uk 


The pollinator community in the UK has been steadily in decline due to a variety of 

reasons including monoculture, climate change, pesticides and diseases. Several honey 

bee diseases have been well studied, although interspecific transmission between 

pollinators has received little attention. In order to assess the occurrence and spill over of 

cryptic parasites in pollinators, we screened pollinators using PCR that were collected 

foraging in and around the Leeds and North Yorkshire area. We screened bumblebees, 

honeybees, wasps, hoverflies and solitary bees. We found several common honeybee 

infections in all pollinators, including Nosema, Wolbachia, and Chalkbrood. We found a 

Nosema infection rate of approximately 7%, with the majority of infections being N. 

bombi, found in the bumble bee Bombus pascuorum, but also across all groups screened. 

These results highlight the need for community level, rather than species level studies 

when considering issues such as pollinator declines.





EFFECTIVENESS IN REDUCING THE NUMBER OF NOSEMA SPORES OF 

API HERB AND VITA FEED GOLD 




Giacomelli A 1 , Ferrari C 2 , Milito M 1 , Muscolini C 1 , Ermenegildi A 1 , Aquilini E 1 , 

Formato G 1* 

1 

Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana 

2 Azienda USL RM/G 

*, Istituto Zooprofilattico Sperimentale delle Regioni Lazio e 

Toscana, Via Appia Nuova, 1411 – 00178 Roma (Italy) 


Api Herb and Vita Feed Gold are liquid feeds that are on sale in EU for the prevention 

and control of Nosemosis. While Api Herb is based on vegetable essences and vitamins, 

Vita Feed gold is based on natural beet extract and molasses. 

In this work we report the results of the effectiveness of the two mentioned products in 

reducing the number of spores of Nosema, after six weeks of treatments. 

Both Api Herb and Vita Feed Gold resulted able to control the Nosema infection, as it 

proven by a statistically significant difference between the untreated group and the two 

treated groups (Bonferroni post-hoc test for multiple comparisons). 

For the first three weeks of treatments, Api Herb and Vita Feed Gold showed a similar 

ability to control the Nosema infection, but from the 3 rd week ahead, the Vita Feed Gold 

treatment seemed to better control the number of spores of Nosema.





The size of bee sample for investigation of Nosema sp. infection level in honey bee 

colony. 




Anna Gajda * 

Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Ciszewskiego 8, 02- 

786 Warsaw, Poland 

anna_gajda@sggw.pl 

Tel.: +48225936140 


OIE recommends two methods for microscopic diagnosis of Nosema infection: nonquantitative 

and standardised. In the first method 60 forager bees should be investigated, 

in the second - 10 bees. Higes with co-authors pointed out that the number of N. ceranae 

spores per bee does not reflect the level of infection because midgut epithelial cells 

contain many more developmental stages of these spores than is the case in N. apis 

infection; therefore real-time PCR or counting infected bees should be performed. For 

this purpose 30 forager bees should be examined. However, in Poland collecting 30 

foragers at some times of the season is often very difficult or almost impossible, e.g. 

between the middle of July and the middle of August, or in early spring if the colony is 

weak. In such cases alternative sampling should be proposed. 

A three stage investigation of samples (composed of 30 forager bees) performed by us 

showed, that if only 20 bees were investigated 7 out of 44 positive samples could be 

found as negative. In 17 out of the 37 remaining samples the percentage of infected bees 

was at least twice as low in one batch of ten bees as in the other batch. Our conclusion is 

that also in Poland 30 foragers should be investigated. 

Examination of samples composed of 20 foragers and samples of 2 x 20 interior bees 

(from the last comb) showed that although the number of spores per bee was much higher 

in the samples of foragers, the percentage of infected bees was almost the same in both 

kinds of samples if we chose the more infected batch of twenty interior bees for 

comparison.





To bee or not to bee: differential mortality induced by Nosema ceranae 




Ulrike Hartmann 1 *, Jean-Daniel Charrière 1 , Marco Lodesani 2 , Peter Neumann 1 

1 Swiss Bee Research Centre, Agroscope Liebefeld-Posieux Research Station ALP, 

Schwarzenburgstrasse 161, CH-3003 Bern, Switzerland 

2 CRA - Unità di Ricerca di Apicoltura e Bachicoltury, Via Fratelli Rosselli, 80, I-4210 

Reggio Emilia (RE), Italy 

* Contact author: hartmann.ulrike@alp.admin.ch, Phone: +41 31 324 74 24 


The endoparasitic microsporidian Nosema ceranae is a major suspect for the recent 

honeybee colony losses but there may be differences because both host and parasite are 

genetically diverse. Therefore, we here studied mortality of freshly emerged honeybee 

workers individually infested with Italian N. ceranae (10 5 spores per bee) using standard 

hoarding cage experiments over a period of 15 days. We compared our data with the 

literature on Chinese N. ceranae (Paxton et al. 2007). Our data revealed neither 

significant differences between the controls nor between N. apis (Paxton et al. 2007) and 

N. ceranae (this study). However, we found significantly lower mortality induced by 

N. ceranae compared to Paxton et al. (2007). Our results therefore suggest that there may 

be differences in host susceptibility and/or N. ceranae virulence, which should be tested 

using standardized COLOSS ring tests.





Mechanisms through which Nosema apis affects onset of foraging 

in worker honeybees (Apis mellifera L.) 




Huaron Lin 1 , Joseph Sullivan 2 , Zachary Y. Huang 1,3* 

1 Department of Entomology, Michigan State University, East Lansing, MI 48824 

2 University of Massachusetts Memorial Hospital, Department of Orthopedics and 

Physical Rehabilitation, 119 Belmont Street, Worcester, MA 01605 

3 Ecology, Evolutionary Biology, and Behavior Program, Michigan State University, 

East Lansing, MI 48824 

* bees@msu.edu, 517-353-8136. 


It is known that Nosema apis accelerates the rate of behavioural development in 

honeybee workers, but the underlying mechanisms are unknown. We first confirmed that 

juvenile hormone titers were higher in Nosema infected bees that were of preforaging 

age. The higher JH titers can be achieved by several alternative mechanisms: enhanced 

JH production by host corpora allata (CA), reduced JH degradation, or JH production by 

Nosema directly. Three experiments were conducted to further study the mode of action 

of Nosema. Nosema infected workers had higher rates of JH biosynthesis than control 

bees in 4 of 4 colonies when workers were 6-8 days old. Rates of in vivo JH biosynthesis 

were also higher in Nosema infected bees than control bees. Allatectomized workers fed 

Nosema had no detectable levels of juvenile hormone in hemolymph. The majority of 

these workers (two out of three colonies) did not show earlier foraging compared to the 

control group (allatectomized bees with no Nosema). Finally, nosema-infected workers 

also showed higher JH degradation compared to control bees. These results suggest that 

Nosema-infected workers forage at an earlier age than control bees due to higher JH 

titers, which come about through increased JH production, and despite of the increased 

JH degradation in infected bees. Our results also suggest that Nosema apis does not 

produce JH directly.





Nosema situation in the Czech republic 




Martin Kamler 1 , Dalibor Titra 2,* , Jan Tyl 2 , Štpán Ryba 2,3 , Betislav Koudela 1, 4 

1 Department of Parasitology, Faculty of Veterinary Medicine, University of Veterinary 

and Pharmaceutical Sciences, Palackého 1/3, 612 42 Brno, Czech Republic 

2 Bee Research Institute Dol, 252 66 Libice nad Vltavou, Czech Republic 

3 Department of Zoology, Faculty of Science, Charles University, Vininá 7, 128 44, 

Praha, Czech Republic 

4 

Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, 

Branišovská 31, 370 05 eské Budjovice, Czech Republic 

* Author for correspondence: beedol@beedol.cz; phone: +420 220 940 480 


The status of nosemosis in honeybee colonies in the Czech Republic is traditionally 

demanded by State Veterinary Administration and investigated in Bee Research Institute 

at Dol (BRI) for many years. The Nosema, Acarapis, Varroa and Paenibacillus 

investigation is obligatory for all commercial queen breeders. 

Method of examination is conducted microscopically, according to OIE recommended 

diagnostic techniques (version 2008) adapted by BRI. Positive result is finding of >10 6 

Nosema spores in disintegrated sample of 20 bees. This finding corresponds to 5 % ill 

bees. 

We summarized results for nosemosis monitoring from the period 2000 – 2009. In this 

period the accredited laboratory of BRI received samples from 76 to 114 queen breeders 

every year. Average number of samples was 3945 per year (minimum 2771 and 

maximum 6142). Microscopically, the mean prevalence of Nosema sp. spores was 29 % 

(24 % - 44 %) in individual year. Because of worldwide occurrence of microsporidian 

pathogens Nosema ceranae together with N. apis, we analyzed 92 Nosema positive 

samples specifically by molecular probes. DNA of N. ceranae was confirmed in 7 

samples (6 %). Occurrence of N. ceranae in honeybee colonies in the Czech Republic 

was confirmed for the first time. However, the role of N. ceranae in colony collapse 

disorder in our apiaries is not known.





NOSEMA DIAGNOSTIC 




R. Martín-Hernández*, C. Botías, A. Meana**, M. Higes* 

*Centro Apícola Regional (CAR). Camino de San Martín s/n, 19180 Marchamalo, Spain. 



Corresponding author: R. Martín-Hernández, rmhernandez@jccm.es 


In order to make a good diagnostic, it is very important to do a good selection of 

sample to analyze, a good establishment of parameters to determine disease or not and 

finally use good detection methods. 

For Nosema apis, the mean spore count per bee has been used to determine the 

extent of infection in a colony (Furgala & Hyser, 1969; Cornejo and Rossi, 1975) and this 

parameter has been even used to evaluate the need to apply treatment in infected colonies 

and apiaries (Sota and Bacci, 2004). However, some authors established that there is not 

a close relationship between this measure and the colony health status (Doull, 1965, El 

Shemy & Pickard, 1989). 

Due to the scarce information for N. ceranae, different trials were made on natural 

infected colonies. These works demonstrate that the election-sample should be exteriorbees 

collected at noon at the hive entrance. On the other hand, spore count is not related 

with parasitic burden and health status of colonies as same authors described previously 

for N. apis (Doull, 1965; El Shemy and Pickard, 1989). Therefore, the best parameter to 

determine the health status of a colony infected with N. ceranae is the percentage of 

infected bees. The detection of infection has been shown with a high influence of the size 

sample that is get from the colony. 

Finally, the use of triplex PCR technique to detect N. apis and N. ceranae infection 

in honeybees is improved by using COI (A. mellifera) as a target to control the DNA 

extraction.





NOSEMA DIAGNOSTIC 




R. Martín-Hernández*, C. Botías, A. Meana**, M. Higes* 

*Centro Apícola Regional (CAR). Camino de San Martín s/n, 19180 Marchamalo, Spain. 



Corresponding author: R. Martín-Hernández, rmhernandez@jccm.es 


In order to make a good diagnostic, it is very important to do a good selection of 

sample to analyze, a good establishment of parameters to determine disease or not and 

finally use good detection methods. 

For Nosema apis, the mean spore count per bee has been used to determine the 

extent of infection in a colony (Furgala & Hyser, 1969; Cornejo and Rossi, 1975) and this 

parameter has been even used to evaluate the need to apply treatment in infected colonies 

and apiaries (Sota and Bacci, 2004). However, some authors established that there is not 

a close relationship between this measure and the colony health status (Doull, 1965, El 

Shemy & Pickard, 1989). 

Due to the scarce information for N. ceranae, different trials were made on natural 

infected colonies. These works demonstrate that the election-sample should be exteriorbees 

collected at noon at the hive entrance. On the other hand, spore count is not related 

with parasitic burden and health status of colonies as same authors described previously 

for N. apis (Doull, 1965; El Shemy and Pickard, 1989). Therefore, the best parameter to 

determine the health status of a colony infected with N. ceranae is the percentage of 

infected bees. The detection of infection has been shown with a high influence of the size 

sample that is get from the colony. 

Finally, the use of triplex PCR technique to detect N. apis and N. ceranae infection 

in honeybees is improved by using COI (A. mellifera) as a target to control the DNA 

extraction.





Histopathology of Nosema disease. 




Aránzazu Meana1*, Pilar Garcia Palencia1, Raquel Martín Hernandez2, Mariano Higes2. 

1 Facultad de Veterinaria, Universidad Complutense de Madrid 

2 Centro Apícola Regional, JCCM, Marchamalo. 


Data presented have been originated in several trials based either in experimental 

infections (nurse honey bee with spores or queens with infected nurses), or in natural 

infection in CAR apiaries and professional apiaries (interior and exterior bees). 

Once refreshed the histology of digestive epithelium of honey bee, description of infected 

cells will be explained based on endogenous biological cycle of Nosema spp. divided in 

phases. 

Phase I is the extracellular stage of Nosema ceranae includes mature spores. Under 

appropriate conditions, the spore is activated in bee gut, its polar filament everts and 

pierces a bee intestinal cell, injecting the sporoplasm into it. Phase II is the first phase of 

intracellular development. Nosema ceranae maintains direct contact between 

plasmalemma and host cell cytoplasm lacking any type of interfacial envelope. Once 

injected, the sporoplasm forms an elongated multinucleate cell that divides by multiple 

fission. Phase III or the sporogonic phase. Material secreted by the parasite forms an 

electro-dense addition to the plasmalemma. Then cell undergoes one nuclear division 

process by binary fission producing two sporoblast cells that mature to produce spores 

Conclusions: All the life cycle stages of Nosema are diplokariotics. All parasitic stages 

are in direct contact with the host cell cytoplasm. Emptied spores probably indicate 

intracellular germination of spores and a quick spreading through epithelial layer. 

Regenerative cells are not activated, alterations are irreversible and a death cause. 

Some of the images to be seen: 

Experimental infection day 3 p.i. A few epithelial cells at the tip of the folds presenting 

intracellular parasite stages, mature spores present. 

Experimental infection day 6 p.i. Heavily infected tissue with tightly packed parasites. 

Basophilic mature spores in apical region of epithelial cells. 

Large heterogeneously stained parasitic structures at the bottom of the folds.Parasitic 

epithelial cells and perithrophic membranes fragments in the gut lumen. Cell nuclei are 

displaced to an apical position. Empty spores clearly indicate horizontal transmission. 

Cellular membrane appears disrupted. Regeneration crypts are not activated.





Epidemiology of Nosema disease. 




Aránzazu Meana1*, Amparo Martínez2, Raquel Martín Hernandez3, Mariano Higes3. 

1 Facultad de Veterinaria, Universidad Complutense de Madrid 

2 Tragsega, Madrid 

3 Centro Apícola Regional, JCCM, Marchamalo. 


Nosemosis is a common worldwide disease of adult honey bees (Apis mellifera) that is 

caused by microsporidia. Nosema apis infecting Apis mellifera was described more than 

one hundred years ago and is one of the first microsporidia to be described. Recently a 

second microsporidian, Nosema ceranae, infecting the same host has been reported in 

Europe and Asia, being considered nowadays also a pandemia. The relative risk obtained 

in colonies with either both species or only N. ceranae, for suffering bee depopulation is 

almost six times greater than those with negative PCR results. Epidemiological patterns 

in Spain for N. ceranae include a long asymptomatic phase previous to cold month’s 

depopulation and colony death. Different factors relate with disease such as type of host, 

temperature influence on parasite biotics and biological effects, or immune deflection of 

N. ceranae infected honeybees compared with N. apis will be presented. 

While the disease caused by N.apis is accepted to be transmitted among bees via 

ingestion being the reservoirs live infected bees that contaminated the combs and deposits 

of viable spores on or in wax, honey or any hive surface, the same is suspected for N. 

ceranae. The rapid, long distance dispersal of N. ceranae has been attributed to the 

transport of infected honey bees by commercial or hobbyist bee keepers but there may be 

other alternatives. Hive structures can transmit the infection. The transmission by means 

of frames and other materials from the continent to an island of the Netherlands has been 

confirmed. The presence of viable spores in corbicular pollen can be considered a high 

spreading factor. The demonstrated viability of spores inside the regurgitated pellets of 

bee-eaters indicates that they can act as fomites of infective spores. The flying behaviour 

of bee-eaters can spread them all over long distances. Mortality and morbility rates are 

discussed while considering a dead colony the absence of a queen and the need to 

establish new parameters to identify nosemosis by N. ceranae due to absence of clinical 

signs related with N. apis. Percentage of forager’s infection is more reliable to show a 

relationship with the impact of infection than mean spore count in a colony infected by N. 

ceranae.





Molecular basis of genome interaction of the honeybee Apis mellifera with an 

evolutionary old and novel introduced Nosema species 




Matthias Müller, Robin FA Moritz* 

Martin-Luther-University Halle-Wittenberg 

r.moritz@zoologie.uni-halle.de 

Hoher Weg 4 

06099 Halle 

+49 345 55 26 223 


The honeybee, Apis mellifera, and two Microsporidian parasites, Nosema apis 

and N. ceranae will be used to study coevolutionary mechanisms of host 

resistance and parasite virulence. N. apis is an evolutionary old and well adapted 

parasite of the honeybee whereas N. ceranae is a novel, highly virulent, and 

potentially mal-adapted parasite of A. mellifera that recently swapped hosts from 

A. cerana. We will use the sequenced genome of A. mellifera and genomic tools 

to take advantage of the haploid drones as simple genetic test organisms in 

evolutionary genetics studies. We will identify major quantitative trait loci 

responsible for resistance to N. ceranae, N. apis and mixed infections testing 

drones, which are offspring of hybrid queens of susceptible and more resistant 

lineages. 

We will compare the transcriptome of drones and workers to asses the impact of 

heterozygosity on parasite resistance, and test for the selection potential of 

virulence and its trade off with transmission ability in Nosema. Finally, we will 

identify between parasite selection within the honeybee host.





Presence of Nosema apis and Nosema ceranae in Italian apiaries 




Anna Granato*, Mauro Caldon, Christian Falcaro, Franco Mutinelli 

NRL for beekeeping, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro 

(Padova) Italy 

Dr.ssa Anna Granato 

Istituto Zooprofilattico Sperimentale delle Venezie 

Viale dell’Università, 10 

35020 Legnaro (Padova) Italy 

Tel. 0039 049 8084259 

Fax 0039 049 8084258 

e-mail: agranato@izsvenezie.it 


The present study describes the results of a preliminary screening to assess the presence 

of Nosema apis/Nosema ceranae in Italian apiaries. 

238 samples of adult honey bees from 19 of the 20 Italian regions (Valle d’Aosta was not 

included) were submitted to the Istituto Zooprofilattico Sperimentale delle Venezie for 

diagnosis of N. apis/N. ceranae infection. Honey bee crushings were subjected to light 

microscope examination (400X) to detect the presence of Nosema spp. spores and to 

DNA extraction by QIAamp DNA mini kit (Qiagen Gmb, Hilden Germany) with a preincubation 

step with lysozime. DNA was amplified by using specific primers for a region 

of 16S rRNA gene of Nosema spp (Higes et al, 2006) and the samples were tested in 

duplicate. For species identification, the PCR products were sequenced and the sequence 

similarity analysis was performed using BLAST database search. 

Of 238 analysed samples, 95 were negative and 143 produced a PCR product of expected 

size, approximately 240-252 bp. After PCR product sequencing, 136/143 samples were 

positive for N. ceranae and in one sample both N. apis and N. ceranae were present. The 

sequence analysis of the other 7 samples identified different species of Nosema. 

Our results confirmed the preliminary findings of Klee et al (2007) on Italian apiaries and 

demonstrated that N. ceranae is a well established parasite of Apis mellifera in all 19 

investigated Italian regions. Furthermore, in only one apiary in Trentino Alto Adige 

region (northern Italy) a co-infection N. apis/N. ceranae was present.





ApiHerb as an alternative product to treat nosema infection. 




Antonio Nanetti 

antonio.nanetti@entecra.it 

CRA-API. Consiglio per la Ricerca e la sperimentazione in Agricoltura. Unità di Ricerca 

di Apicoltura e Bachicoltura 

Via di Saliceto 80, 40128 Bologna, Italia 

Tel.: ++39 051 353103 


Nosema apis and N. ceranae are causative agents of two different forms of nosema 

disease. In particular, the latter microorganism is claimed as a major factor of 

depopulation and death in Apis mellifera colonies in regions where beekeeping is 

performed at economic level. 

Fumagillin was once generally available as an effective medication, but regulatory 

restrictions banned it from several countries where, aside illegal import and use, the 

control means are restricted to higienic practices, usually of unadequate efficacy. 

In this context, the product ApiHerb (Chemicals Laif, Padua, Italy), based on herbal 

extracts, was developed and tested. 

In laboratory, uninfected caged honey bees were fed with the substance as a syrup 

suspension (ad libitum for one day) and artificially infected with N. ceranae spores. The 

individual spore load of the bees was evaluated 10 days post-infection, recording 

significantly milder infections if compared to untreated controls. 

Free flying colonies of a severely hit apiary received three weekly administrations of the 

product by trickling. The pre - post treatment difference of luminal spores was measured 

in bee samples, resulting in a significant decrease (-46%) if compared to negative 

controls. The decrease was not significantly different to the one recorded in positive 

controls (-60%) receiving fumagillin two times, one week apart. 

The experiments show a distinct effect of ApiHerb in limiting the development of N. 

ceranae infection of the honey bees. Due to its herbal composition, the product may suit 

the restrictions of the organic beekeeping also.





Physiological and behavioural changes in Nosema infected bees: A model to understand 

colony collapse. 




Dr. Dhruba Naug. 

Assistant Professor, Colorado State University, 1878 Campus Delivery, Fort Collins, CO 

80523, U.S.A. 

dhruba.naug@colostate.edu 


In spite of the tremendous interest in the recent large honeybee losses attributed to colony 

collapse disorder, there is still no definitive explanation for the phenomenon. A host of 

pathogens has been implicated in the process including the newly discovered 

microsporidian species, Nosema ceranae. Results from our experiments show that N. 

ceranae exerts a significant energetic stress on its host. This energetic stress leads to a 

number of physiological effects in the host such as an increased hunger level and an 

inability to thermoregulate, which in turn lead to behavioral changes such as a lower 

inclination to share food, a higher propensity to forage and a preference for inhabiting the 

central regions of the colony. These changes have a profound effect on the transmission 

of the disease inside the colony as well as the mortality of bees outside. I argue that 

energetic stress in the host is a general physiological effect of a number of pathogenic 

infections and that energetic stress exacerbated by habitat loss has played an important 

role in the recent colony collapses.





Diversity and recombination of rDNA in the microsporidian Nosema ceranae: how 

reliable is the genotyping 




Nuno Henriques-Gil, Genetics Laboratory, San Pablo-CEU University, Montepríncipe, 



The rDNA sequences are usually one of the first options for genetic markers in a wide 

range of materials. However, in Nosema ceranae overlapping sequences are 

systematically obtained, precluding clear genotyping of different isolates. The results 

suggest that the repeats of the rDNA are not identical within a given strain. We cloned 

rDNA fragments from N. ceranae of different geographical origins in bacterial plasmids. 

A high diversity was obtained among the 105 sequences analysed, that could be grouped 

into 79 haplotypes. While a same haplotype can appear from different isolates (even from 

samples as far as Guadalajara and Kyrgyzstan), two different clones from a same isolate 

may be deeply different. Therefore epidemiological or phylogenetic relationships among 

isolates of N. ceranae, based on repeated sequences such as rDNA, have to be taken with 

caution. Additionally, the comparison of sequences indicates that recombination is 

generating new variants





Epidemyology and Treatment of Nosemosis in Turkey 




Aslı Özkırım* 

Hacettepe University Department of Biology Bee Health Laboratory 06800 Beytepe- 

Ankara/TURKEY 

Author of correspondance: ozkirim@hacettepe.edu.tr 


Nosemosis is a common honeybee disease in Turkey. Even though it is very simple to 

determine the occurrence of the disease in every region of Turkey, infection level is very 

different in some regions. Turkey has 7 geographical regions have different climatic and 

physical conditions. Several samples are collected twice from each region of Turkey 

every year for monitoring Nosema situation in Turkey. Our results show that Nosema 

spores can be detected in all regions but the main difference is infection level of this 

disease. When comparing of the graphs of infection level especially with climatic 

conditions, the maximum number of nosema spores can be found the north of Turkey; 

Black Sea region. (The situation of Nosemosis in other regions will be explained in the 

presentation) 

For treatment, Fumagilline shouldn’t be used legally but most of the beekeepers still use 

this compound by informal way. Depend on infection level, they prefer to apply cultural 

methods to prevent spreading of the disease or to protect their colonies. On the other 

hand, our reseachs are concentrated on botanical compounds for treatment of Nosemosis.





Rare Nosema infections in Denmark 




Per Kryger * 

per.kryger@agrsci.dk 

University of Aarhus, Faculty of Agricultural Sciences, Dept. of Integrated Pest 

Management, Forsøgsvej 1, DK-4200 Slagelse, Denmark 

Phone: +45 8999 3629 


In Denmark beekeepers have been trying to produce Nosema resistant bees for the past 

nearly 20 years. Today we have far less problems with Nosema, compared to when we 

started. In general beekeepers think the program has been a success. There might be other 

explanations for the decrease in Nosema cases. Using more hygienic methods when 

cleaning hives the beekeepers, having isolated and ventilated hives, and working mainly 

from stock that is free of Nosema when splitting colonies all contributes to reduce 

Nosema in apiaries. Still, the queen breeders of Denmark would be very keen to have 

their bees tested, both for Nosema apis and Nosema ceranae. They presently rely on the 

counting Nosema spores from breeder colonies in early spring only. The better way 

would be to expose bees to Nosema infections in lab, and see if indeed there is a 

difference. 

The Danish work has been undertaken entirely by private breeders. The beekeepers have 

been rather smart, and already nearly 20 years ago developed a computer assisted 

Nosema spore counting system, which seems to work very well for them. I will give an 

overview of the methodology used, and the results achieved. The first record of Nosema 

ceranae is from 2005, but probably the new Nosema arrived well before that in Denmark 

given the place of the first discovery.





Genetic variation in resistance to Nosema infection within honeybee colonies 




Katherine Roberts 1 , Giles Budge 2 & William Hughes 1* 

1 


2 The Food and Environment Research Agency, UK 

* Author for correspondence: 

Institute if Integrative and Comparative Biology 

Faculty of Biological Sciences 

Miall Building 


Leeds 

LS2 9JT 

UK 

Phone: +44 (0)113 3437214; Email: w.o.h.hughes@leeds.ac.uk 


In the UK, current nosemosis infection is due to both Nosema apis and Nosema ceranae. 

Originally N. apis was the sole causative agent of the disease, however increasingly N. 

ceranae is found to be co-infecting or displacing N. apis. Honeybee colonies are 

genetically diverse due to the multiple mating of queens, leading to genetic variation in 

the workers making up a colony. This variation may also lead to variation in resistance 

to parasites, and further complicate the host-parasite interaction of Nosema with differing 

host genotypes. Understanding the host-parasite dynamics of infections is crucial to 

detangling the pathology and control of both Nosema species. In this study we are 

examining the effect of host genotype and parasite species on nosemosis. We found 

using quantitative PCR that most natural infections were mixed, meaning production of 

pure Nosema cultures was needed for controlled experimentation. We then used these 

pure cultures of both Nosema species to experimentally infect individual workers of 

Nosema free colonies. These individuals were then checked for infection using both 

spore counts and quantitative real-time PCR, and later genotyped, in order to look for 

variation in infection of workers based on their patriline. This study determines if 

honeybees genotypically vary in resistance to both Nosema species, and could feed back 

into the apicultural industry to help breed more resistant lines of honeybee.





First detection of Nosema ceranae in Apis mellifera from 

Bosnia and Herzegovina 




* Violeta Santrac, Veterinary Institute of Republic of Serpska, Banja Luka, Bosnia and 

Herzegovina, santracv@veterinarskiinstitutrs.com 

Anna Granato, Franco Mutinelli, Istituto Zooprofilattico Sperimentale delle Venezie, 

National reference laboratory for beekeeping, Legnaro (Padova), Italy 


Many neighbouring countries have reported high prevalence of Microsporidia and 

its different epidemiological patterns. 

The aim of this work was to gain knowledge about the presence of N. ceranae in 

Bosnia and Herzegovina’s honey bees, its implication on bee pathology, and future needs 

for the more efficient diagnostic molecular tools. 

• Fifteen samples of dead honey bees were analyzed according to the OIE Manual 

(OIE, 2008) for Nosema disease. Levels of spores in naturally infected bees were 

different and scored as low, middle, and very high spore count. 

Samples were collected during February and March of 2008 and 2009 from randomly 

selected areas of seven epizootiological units in Bosnia and Herzegovina (Prijedor, 

Zvornik, Gradiska, Doboj, Banja Luka, Modrica, Dubica 

Since molecular diagnostic of Nosema species does not currently exist in Bosnia and 

Herzegovina, we asked the Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro 

(Padova, Italy) to perform this diagnostics as part of COLOSS project cooperation. 

Results 

1. All fifteen samples were positive for N. ceranae 

2. Neither N. apis alone nor N. apis/N. ceranae co-infection were detected

Nosema Disease: Lack of Knowledge and Work ... - COLOSS

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